Conformation-dependent cleavage of hairpin and triplex nucleic acids by a temperature-insensitive photonuclease

Photolysis of Pt-2(pop)(4)(4-) in the presence of duplex DNA produces strand scission via abstraction of the 4'- and 5'-hydrogen atoms (pop = P2O5H22-). The cleavage intensities are higher for single-stranded DNA compared to those for duplex DNA because of lower electrostatic repulsion and greater solvent accessibility in single strands. When the single-stranded oligomer d(5'-ACTGCCTTTTTGCTGAA) was. photolyzed in the presence of Pt-2(pop)(4 )(4-), there was no significant or systematic change in cleavage intensity as a function of temperature in the range 28 to 98 degrees C. Cleavage of the hairpin d(5'-ATCCTATTTTTTAGGAT) showed a higher cleavage intensity in the 5'-TTTTT loop region compared to that for the base-paired nucleotides. Thermal denaturation of the hairpin gave a less selective cleavage pattern; The increase in cleavage at the duplex nucleotides could be used to estimate the T-m,for the folded hairpin; thus, the cleavage pattern reflects the thermal denaturation of individual nucleotides. This concept was tested on the oligomer d(5'-GAAGAGGTTTTTCCTCTTCTTTTTCTTCTCC), which exists in either a single-, double-, or triple-stranded form, depending on the pH and temperature. The folded forms exhibit higher reactivity in the loop regions compared to that for the base-paired nucleotides. Thermal denaturation of the base-paired nucleotides could be distinguished in the cleavage patterns and correlated with conventional optical absorption data. While optical melting curves provide greater precision in the observed T-m, values, the cleavage intensity approach allows mapping of multiple transitions to individual nucleotides in the-sequence. The combination of both approaches therefore offers a powerful method for following thermal denaturation of DNA at A, T, and C nucleotides.